Platelets suppress T_reg recruitment

Apart from their canonical role in thrombosis and hemostasis, platelets amplify acute and chronic inflammation including atherosclerosis, and regulate the development and remodeling of blood and lymph vessels in health and disease. In this issue of Blood, Lievens and colleagues show evidence that activated platelets expressing CD40L promote atherogenesis and inflammation at least in part by mitigating the natural response to recruit immunomodulatory T_regs.¹ 

Mounting evidence indicates a pivotal role for regulatory T cells (T_regs; generally identified by positive expression of CD4, CD25, and FoxP3) in balancing inflammation and autoimmune disease via actively suppressing pathogenic immune activation (Th1, Th2, Th17, CTL) and maintaining immune tolerance.²  Although present approximately 1% to 2% in whole blood, they are actively recruited to inflammatory sites via various chemokines including CCL2 and CCL5.²  In addition, in atherosclerosis (ie, a chronic inflammatory disease of the vessel wall caused by progressive accumulation of lipids), various groups have shown an inverse relationship between the presence of T_regs versus atherosclerotic plaque growth, and even the induction of a “stable plaque” phenotype characterized by reduced inflammatory cell infiltration and increased plaque fibrosis.^3,4  The latter is clinically important, as such stable plaques would prevent plaque rupture thereby reducing the associated life-threatening risk of thrombo-occlusive/ embolic stroke and myocardial infarction.⁵ 

CD40 ligand (CD40L; CD154) is a transmembrane glycoprotein and member of the tumor necrosis factor (TNF) family. While existing in a membrane-bound and soluble form, CD40L can bind upon trimerization to its classical receptor CD40 leading—via TNF receptor–associated factor (TRAF) adaptor proteins—to intracellular signaling and transcriptional activation, but CD40L also signals by binding noncanonical receptors including integrins (reviewed in Seijkens et al⁶ ). Primarily known for its role as a costimulator of T-cell activation, it became clear that CD40L is also functionally expressed on several other hematopoietic and nonhematopoietic cell types including platelets and endothelial cells.⁶  In atherosclerosis, various groups showed that inhibition or genetic deletion of CD40L in mice reduced the growth of newly formed as well as established plaques, and promoted the acquisition of a stable plaque phenotype.^7,8  Given the broad expression of CD40L on several cell types relevant in atherosclerosis, it seems important to identify the proatherogenic role of CD40L in a cell-type–specific manner.

Here, Lievens and colleagues demonstrate, in 3 laborious mouse models of atherosclerosis (early/advanced/established lesions), that repeated transfer of thrombin-activated platelets doubles plaque size in all phases, and this phenomenon is strictly dependent on CD40L expression on platelets (evidenced by using CD40L^−/− platelets) in combination with the presence of natural T_regs (evidenced by using anti-CD25–depleting antibodies).¹  Thus, the absence of platelet CD40L increases the number of circulating T_regs, thereby blocking plaque growth induced by activated platelets (see figure). Surprisingly, the authors found that activated CD40L⁺ platelets increased—not reduced—the plasma levels and endothelial deposition of CCL2, while CCL5 expression was not regulated by platelet CD40L.¹  Therefore, the inverse relationship between platelet CD40L versus T_regs does not appear to be explained by unfavorable chemokine gradients. In addition, T_reg function seemed unaltered as corroborated by in vitro assays and normal IL-10 plasma levels.¹ 

Interestingly, Lutgens et al recently reported that the absence of leukocytic TRAF2/3/5 signaling downstream of CD40 also increased T_reg counts; yet, atherogenesis was unaffected (but no activated platelets were administered).⁹  These findings together suggest that interactions between activated CD40L⁺ platelets and CD40⁺ T_regs might result in TRAF2/3/5 signaling and suppress T_reg recruitment. Hence, apart from distinct tissue-derived chemokine gradients,²  T_reg trafficking into inflamed tissues might be regulated by activated platelets. It would perhaps be interesting to study whether (1) this interaction occurs after solubilization via rapid proteolytic shedding from activated platelets (eg, by ADAM10) or via CD40L⁺ microparticles¹⁰ ; (2) membrane-bound CD40L initiates heterotypic cell-cell interactions with CD40⁺ T_regs; and (3) CD40L-CD40 interaction results in reduced T_reg formation via suppressing FoxP3 and/or CD25 signaling, or increased T_reg apoptosis and/or clearance via overstimulation (see figure).

Consistent with previous observations,¹¹  the authors show that, overall, activated CD40L⁺ platelets only modestly affect plaque composition, with small and inconsistent differences in macrophage infiltration and fibrosis.¹  Hence, platelet CD40L does not seem to primarily promote plaque instability, as suggested by CD40L inhibition/deletion studies.^7,8  Notably, activated CD40L⁺ platelets also did not alter the cytokine release and phagocytic activity of macrophages,¹  and a recent report indicated that platelet CD40L was not required to cross-activate (noninflammatory) endothelium in vivo.¹²  Instead, the current report shows that platelet CD40L promoted the endothelial translocation of themselves, and of leukocytes in vivo—consistent with the increased CCL2 expression and endothelial deposition (see above).¹  In addition, activated CD40L⁺ platelets formed more (proinflammatory) aggregates with each other and with (CD40⁺) leukocytes.¹  Together, these results further underscore the premise that a complex union (a sort of thrombotic “ménage-à-trois”) among platelets, endothelial cells, and leukocytes strongly catalyzes acute and chronic inflammation.¹³  In fact, when considering that activated CD40L⁺ platelets strongly promote intraluminal interactions among platelets, endothelial cells, and leukocytes—including suppressing the circulating number of T_regs, whereas they, at best, modestly promote the intraplaque infiltration of inflammatory (and vascular smooth muscle) cells—one could speculate the following (see figure): analogous to the tightly regulated pro- versus antithrombotic balance, the endothelial cell surface can dynamically shift between pro- versus anti-inflammatory states. On atherosclerotic plaques, activated CD40L⁺ platelets might form some kind of “proinflammatory cell coating” at the luminal side of the intimal endothelial cells which consists of activated leukocytes, platelets, and platelet-leukocyte aggregates. This plaque-specific cell coating tips the balance (via multiple cellular and—as yet incompletely defined—humoral interactions) in a positive amplification loop toward persistent proinflammation, while it concomitantly shields the growing plaque from potentially plaque-growth–inhibiting effects such as the presence of T_regs (ie, reducing their circulating number, via unidentified mechanisms). In a second phase, this proinflammatory balance might further promote plaque growth by increased neovascularization¹⁰  and capillary rarefaction (as evidenced in the current study by the aberrant erythrocyte/iron deposits¹ ).

Finally, this report¹  does not provide evidence that blocking CD40L⁺-activated platelets might prevent atherosclerosis, but it does reinforce earlier clinical findings that antiplatelet drugs including aspirin (typically prescribed to cardiovascular patients) might, apart from preventing thrombotic complications after plaque rupture, also suppress plaque growth.¹⁴  Although the authors as well as previous studies already highlighted that blocking platelet CD40L might reduce thrombus formation and stability,^1,15  the current report identifies—for the first time—that blocking platelet CD40L might preserve the natural T_reg response, as a potential novel mechanism to explain the putative plaque-growth–suppressing effects of antiplatelet drugs.